Various diagnostic instruments are known which are used in the detection of infectious disease agents and antibodies thereto. One such instrument uses a microtiter-plate based enzyme linked immunoabsorbant assay (ELISA). The development of microtiter-plate ELISA methods allows the processing of a large number of samples (typically 96 wells per plate) simultaneously. A common problem in the use of the microtiter-plate based ELISA method is the tracking of sample identity. The wells in a microtiter plate are closely spaced. When a blood sample is manually transferred from a sample tube to one of the closely spaced wells, it can easily be misplaced into a neighboring well.
Several laboratory and robotic systems have been developed for the purpose of processing microtiter plates. These devices are designed to increase laboratory throughput and many of these devices also provide positive sample identification through the use of barcode labels.
For example, the Zymate robot (Zymark Corporation, Hopkinton, Mass., U.S.A.) has been adapted for the processing of the microtiter plates in the research laboratory. Another laboratory automation device that has been in use in many clinical laboratories is the Hamilton MICROLAB-AT (Hamilton Corporation, Reno, Nev., U.S.A.).
The Hamilton MICROLAB-AT, which is also supplied by DuPont as an OEM equipment known as the SUMMIT.TM., is capable of performing the following functions:
1) Reading the barcode labels on each of the 96 sample tubes placed in a tube rack.
2) Adding a precisely measured amount of reagent to each of the 96 wells on the microtiter plate.
3) Transferring a precisely measured amount of sample from the sample tube to the corresponding well on the microtiter plate.
4) Mixing the reagent with the sample and performing any dilution, if required.
In order to minimize the hazards involved in the transfer of human serum which may contain infectious disease agents, the DuPont SUMMIT uses disposable pipets which are ejected into a biohazardous bag after each run. The use of disposable pipets, however, makes the verification of sample accuracy and precision more difficult.
The relatively small size of the wells on the microtiter plate (typically 300 uL per well) requires the precise delivery of a minute amount of sample (typically 10 uL or less). Inaccuracies in the delivery of samples will lead to erroneous results that may endanger public health and the safety of blood supplies. In order to safeguard the accuracy of the ELISA results, the volume delivered by the automated devices must be routinely verified.
Typically, the verification of the volumetric accuracy of a device is accomplished by weighing a sample of pure water transferred by the device. Although this method is a satisfactory procedure for the verification of the delivery of relatively large sample sizes (100 uL or more, i.e. 0.1 g in weight), it is not a satisfactory method for the verification of volumes in the range of 5-10 uL. Sample sizes in the range of 5-10 uL are often required for many microtiter-plate based ELISA methods.
When attempts are made to weigh a 10 uL (or 0.01 g) sample of pure water, the surface tension of water will often lead to the incomplete transfer of the liquid from the pipet tip to the microtiter plate. Moreover, evaporative loss will often lead to errors in the measurment. In order to prevent evaporative loss, the relative humidity of the room must be kept very high. In addition, a highly accurate analytical balance must be used for this kind of measurement.
The proper type of equipment and operating environment which are needed to make gravimetric verification of automated pipetting devices are usually not available in a clinical laboratory. Even if these equipment were available, the skill level and time required to conduct these measurements are very high. Thus, the gravimetric verification of the volumetric accuracy of the robotic ELISA processing devices is not practical in a clinical laboratory.
Typically, in the prior art, colorimetric reagents have been used to verify the volume delivered by a liquid transfer device. In these cases the volume of the reagent is usually calculated by the following equation. (Beer's Law): EQU A=abc
where
A=absorbance measured PA1 a=molar absorptivity PA1 b=path length (usually fixed for a given instrument) PA1 c=concentration of colorimetric reagent.
Using this common approach, the measured volume is dependent on the absolute concentration of the reagent. Therefore, the approach requires that the exact concentration of the reagent is known.
In reality the concentration of the colorimetric reagent, cannot be precisely controlled. Therefore, verification methods that calculate volume directly from the absorbance measured are often inaccurate. Another verification method is justified in U.S. Pat. No. 4,354,376 issued to Greenfield et al. According to Greenfield et al, "In carrying out the invention, a kit is provided comprising a vial of a standard calibration solution of a first predetermined color density, a vial of a standard calibration solution of a third predetermined color density, a calibration vial containing a predetermined volume of a diluent, and one or more vials or bottles, each containing a solution of a calibration reagent of a different color density. The kit may also contain a pipette calibration chart on which spectrophotometer readings of the light adsorption by the solutions in the standard calibration vials are plotted to provide a reference curve can be used to determine the volume of the calibration reagent pipetted into a calibration vial to replace an equal volume of diluent removed therefrom. That volume is the volume of the pipette being calibrated".
The difficulty with Greenfield et al is a manual procedure apparently used for the occasional pipette that needs to be calibrated. It is not at all suitable for a method that contemplates calibrating an automated device.